Specific Developmental Stages Research Articles

Specialized Ribosomes in Health and Disease.

Ribosomal heterogeneity exists within cells and between different cell types, at specific developmental stages, and occurs in response to environmental stimuli. Mounting evidence supports the existence of specialized ribosomes, or specific changes to the ribosome that regulate the translation of a specific group of transcripts. These alterations have been shown to affect the affinity of ribosomes for certain mRNAs or change the cotranslational folding of nascent polypeptides at the exit tunnel. The identification of specialized ribosomes requires evidence of the incorporation of different ribosomal proteins or of modifications to rRNA and/or protein that lead(s) to physiologically relevant changes in translation. In this review, we summarize ribosomal heterogeneity and specialization in mammals and discuss their relevance to several human diseases.

Характеристика факторов внешней среды, влияющих на устойчивость промышленного предприятия

The problem of ensuring the sustainability of the development of an industrial enterprise is a consequence of the rapid pace of changes in the external environment and the needs of business in purposeful and sound strategic management of sustainable development. In an unstable environment, standard management methods and techniques based on standard economic indicators cannot be used to make decisions regarding the sustainable development of an industrial enterprise, which requires new approaches to the study of its sustainability factors. These circumstances determine the scientific and practical significance of studies of environmental factors influencing the sustainability of industrial enterprises. As you know, the sustainability of an industrial enterprise largely depends on the specific stage of business development. Taking into account these conditions, a study of modern approaches to systematization of the factors of influence of an unstable external environment on the stability of an industrial enterprise was conducted. The scientific and practical significance of such studies for improving the effectiveness of strategic sustainability management of an industrial enterprise is substantiated and the conditions for their implementation are determined. Using critical analysis, the results of a number of approaches to the grouping and characterization of environmental factors of enterprises are systematized. Conclusions are drawn about the need to take into account the factors of influence of the external environment of an industrial enterprise in the studies of their interrelations with the factors of the internal environment. The results of the study provide an increase in knowledge in the field of theory and methodology of strategic management of sustainable development of industrial enterprises in the conditions of uncertainty of the external environment. and they are of applied importance for the practice of strategic management, forming the necessary basis for improving the classification of environmental factors influencing the sustainability of an industrial enterprise as one of the tools for strategic decision-making. Classification allows not only to systematize factors for certain reasons, but also to develop an action program to reduce the impact of identified negative factors on the sustainability of the enterprise, taking into account external trends in economic, political, social and environmental development, etc.

AGPs as molecular determinants of reproductive development.

Morphogenesis occurs through accurate interaction between essential players to generate highly specialized plant organs. Fruit structure and function are triggered by a neat transcriptional control involving distinct regulator genes encoding transcription factors (TFs) or signalling proteins, such as the C2H2/C2HC zinc-finger NO TRANSMITTING TRACT (NTT) or the MADS-box protein SEEDSTICK (STK), which are important in setting plant reproductive competence, feasibly by affecting cell wall polysaccharide and lipid distribution. Arabinogalactan proteins (AGPs) are major components of the cell wall and are thought to be involved in the reproductive process as important players in specific stages of development. The detection of AGPs epitopes in reproductive tissues of NTT and other fruit development-related TFs, such as MADS-box proteins including SHATTERPROOF1 (SHP1), SHP2 and STK, was the focus of this study. We used fluorescence microscopy to perform immunolocalization analyses on stk and ntt single mutants, on the ntt stk double mutant and on the stk shp1 shp2 triple mutant using specific anti-AGP monoclonal antibodies. In these mutants, the expression levels of selected AGP genes were also measured by quantitative real-time PCR and compared with the respective expression in wild-type (WT) plants. The present immunolocalization study collects information on the distribution patterns of specific AGPs in Arabidopsis female reproductive tissues, complemented by the quantification of AGP expression levels, comparing WT, stk and ntt single mutants, the ntt stk double mutant and the stk shp1 shp2 triple mutant. These findings reveal distinct AGP distribution patterns in different developmental mutants related to the female reproductive unit in Arabidopsis. The value of the immunofluorescence labelling technique is highlighted in this study as an invaluable tool to dissect the remodelling nature of the cell wall in developmental processes.

N6-methyladenosine reader YTHDF family in biological processes: Structures, roles, and mechanisms.

As the most abundant and conserved internal modification in eukaryote RNAs, N6-methyladenosine (m6A) is involved in a wide range of physiological and pathological processes. The YT521-B homology (YTH) domain-containing family proteins (YTHDFs), including YTHDF1, YTHDF2, and YTHDF3, are a class of cytoplasmic m6A-binding proteins defined by the vertebrate YTH domain, and exert extensive functions in regulating RNA destiny. Distinct expression patterns of the YTHDF family in specific cell types or developmental stages result in prominent differences in multiple biological processes, such as embryonic development, stem cell fate, fat metabolism, neuromodulation, cardiovascular effect, infection, immunity, and tumorigenesis. The YTHDF family mediates tumor proliferation, metastasis, metabolism, drug resistance, and immunity, and possesses the potential of predictive and therapeutic biomarkers. Here, we mainly summary the structures, roles, and mechanisms of the YTHDF family in physiological and pathological processes, especially in multiple cancers, as well as their current limitations and future considerations. This will provide novel angles for deciphering m6A regulation in a biological system.

Histopathological growth pattern evolution of tumor in VX2 liver cancer model

The histopathological growth pattern (HGP) is a morphological reflection of interactions between cancer cells and the surrounding tissue, and has been identified with a remarkably predictive value in liver metastases. However, there is still a lack of studies on HGP of primary liver cancer even furtherly on HGP evolution. We employed VX2 tumor-bearing rabbits as the primary liver cancer model of which tumor size and distant metastasis were investigated. HGP assessment and computed tomography scanning was performed in four cohorts of different time points to map the HGP evolution. Additionally, Fibrin deposition and neovascularization were evaluated by Masson staining and immunohistochemical analysis of CD31, hypoxia-inducible factor-1 alpha (HIF1A) and vascular endothelial growth factor (VEGF). Tumors displayed exponential growth in the VX2 liver cancer model, but these tumor-bearing animals did not show any visible metastasis until they reached a specific stage of development. Correspondingly, the components of HGPs changed along with the tumor growth. The proportion of desmoplastic HGP (dHGP) decreased initially and then grew, but in contrast, the level of replacement HGP (rHGP) rose from the 7th day, reached a peak at around the 21st day, and then appeared drop. Importantly, the collagen deposition and expression of HIF1A and VEGF correlated with dHGP, while CD31 did not. HGP evolution presents a two-way switch including dHGP to rHGP and rHGP to dHGP, in which the emergence of rHGP may be linked to metastases. HIF1A-VEGF partially participates in the HGP evolution and presumably plays a key role in the formation of dHGP.

Root exudate concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) affect maize rhizobacterial communities at specific developmental stages.

Root exudates shape the rhizosphere microbiome, but little is known about the specific compounds in root exudates that are important. Here, we investigated the impacts of the plant-synthesized phytohormones indole-3-acetic acid (IAA) and abscisic acid (ABA) exuded by roots on the maize rhizobacterial communities. To identify maize genotypes that differed in the root exudate concentrations of IAA and ABA, we screened hundreds of inbred lines using a semi-hydroponic system. Twelve genotypes with variable exudate concentrations of IAA and ABA were selected for a replicated field experiment. Bulk soil, rhizosphere, and root endosphere samples were collected at two vegetative and one reproductive maize developmental stage. IAA and ABA concentrations in rhizosphere samples were quantified by liquid chromatography-mass spectrometry. The bacterial communities were analyzed by V4 16S rRNA amplicon sequencing. Results indicated that IAA and ABA concentrations in root exudates significantly affected the rhizobacterial communities at specific developmental stages. ABA impacted the rhizosphere bacterial communities at later developmental stages, whereas IAA affected the rhizobacterial communities at the vegetative stages. This study contributed to our knowledge about the influence that specific root exudate compounds have on the rhizobiome composition, showing that the phytohormones IAA and ABA exuded by roots have a role in the plant-microbiome interactions.

Fine mapping spatiotemporal mechanisms of genetic variants underlying cardiac traits and disease

The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leverage spatiotemporal information on 966 RNA-seq cardiac samples and perform an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identify 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci are enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identifies 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.

Detailed chemical analysis of honey bee (Apis mellifera) worker brood volatile profile from egg to emergence.

Chemical communication is a widely used mode of communication for social insects and has been demonstrated to be involved in many behaviours and physiological processes such as reproduction, nutrition or the fight against parasites and pathogens. In the honey bee, Apis mellifera, the release of chemical compounds by the brood plays a role in worker behaviour, physiology, and foraging activities and colony health as a whole. Several compounds have already been described as brood pheromones, such as components of the brood ester pheromone and (E)-β-ocimene. Several other compounds originating from diseased or varroa-infested brood cells have been described as triggering the hygienic behaviour of workers. So far, studies of brood emissions have focused on specific stages of development and little is known about the emission of volatile organic compounds by the brood. In this study, we investigate the semiochemical profile of worker honey bee brood during its whole developmental cycle, from egg to emergence, with a specific focus on volatile organic compounds. We describe variation in emissions of thirty-two volatile organic compounds between brood stages. We highlight candidate compounds that are particularly abundant in specific stages and discuss their potential biological significance.

Heat stress responses vary during floret development in European spring barley cultivars.

The Poaceae, or grasses, include many agriculturally important cereal crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Barley is a widely grown cereal crop used for stock feed, malting and brewing. Abiotic stresses, particularly global warming, are the major causes of crop yield losses by affecting fertility and seed set. However, effects of heat stress on reproductive structures and fertility in barley have not been extensively investigated. In this study we examined three commercial European spring barley varieties under high temperature conditions to investigate the effects on floret development. Using a combination of fertility assays, X-ray micro computed tomography, 3-dimensional modelling, cytology and immunolabelling, we observed that male reproductive organs are severely impacted by increased temperature, while the female reproductive organs are less susceptible. Importantly, the timing of stress relative to reproductive development had a significant impact on fertility in a cultivar-dependent manner, this was most significant at pollen mitosis stage with fertility ranged from 31.6-56.0% depending on cultivar. This work provides insight into how heat stress, when applied during male pollen mother cell meiosis and pollen mitosis, affects barley fertility and seed set, and also describes complementary invasive and non-invasive techniques to investigate floret development. This information will be used to identify and study barley cultivars that are less susceptible to heat stress at specific stages of floral development.

On the Variability of Software Engineering Needs for Deep Learning: Stages, Trends, and Application Types

The wide use of Deep learning (DL) has not been followed by the corresponding advances in software engineering (SE) for DL. Research shows that developers writing DL software have specific development stages (i.e., SE4DL stages) and face new DL-specific problems. Despite substantial research, it is unclear how DL developers’ SE needs for DL vary over stages, application types, or if they change over time. To help focus research and development efforts on DL-development challenges, we analyze 92,830 Stack Overflow (SO) questions and 227,756 READMEs of public repositories related to DL. Latent Dirichlet Allocation (LDA) reveals 27 topics for the SO questions where 19 (70.4%) topics mainly relate to a single SE4DL stage, and eight topics span multiple stages. Most questions concern <i>Data Preparation</i> and <i>Model Setup</i> stages. The relative rates of questions for 11 topics have increased, for eight topics decreased over time. Questions for the former 11 topics had a lower percentage of accepting an answer than the remaining questions. LDA on README files reveals 16 distinct application types for the 227k repositories. We apply the LDA model fitted on READMEs to the 92,830 SO questions and find that 27% of the questions are related to the 16 DL application types. The most asked question topic varies across application types, with half primarily relating to the second and third stages. Specifically, developers ask the most questions about topics primarily relating to <i>Data Preparation</i> (2nd) stage for four mature application types such as <inline-formula><tex-math notation="LaTeX">${{\sf Image\ Segmentation}}$</tex-math></inline-formula> , and topics primarily relating to <i>Model Setup</i> (3rd) stage for four application types concerning emerging methods such as <inline-formula><tex-math notation="LaTeX">${{\sf Transfer\ Learning}}$</tex-math></inline-formula> . Based on our findings, we distill several actionable insights for SE4DL research, practice, and education, such as better support for using trained models, application-type specific tools, and teaching materials.

Large-scale proteomic analysis of T. spiralis muscle-stage ESPs identifies a novel upstream motif for in silico prediction of secreted products

The Trichinella genus contains parasitic nematodes capable of infecting a wide range of hosts including mammals, birds and reptiles. Like other helminths, T. spiralis secretes a complex mixture of bioactive molecules capable of modulating its immediate surroundings and creating a hospitable environment for growth, survival and ultimately transmission. The constitution of these excretory-secretory products (ESPs) changes depending on the tissue niche and the specific stage of parasite development. Unique to T. spiralis is a true intracellular stage wherein larvae develop inside striated myotubes. Remarkably, the parasite larvae do not destroy the host cell but rather reprogram it to support their presence and growth. This transformation is largely mediated through stage-specific secretions released into the host cell cytoplasm. In this study, we apply state of the art proteomics and computational approaches to elucidate the composition and functions of muscle-stage T. spiralis ESPs. Moreover, we define a recurring, upstream motif associated with the stichosome, the main secretory organ of this worm, and can be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages.

Multimodal human thymic profiling reveals trajectories and cellular milieu for T agonist selection.

To prevent autoimmunity, thymocytes expressing self-reactive T cell receptors (TCRs) are negatively selected, however, divergence into tolerogenic, agonist selected lineages represent an alternative fate. As thymocyte development, selection, and lineage choices are dependent on spatial context and cell-to-cell interactions, we have performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and spatial transcriptomics on paediatric human thymu​​s. Thymocytes expressing markers of strong TCR signalling diverged from the conventional developmental trajectory prior to CD4+ or CD8+ lineage commitment, while markers of different agonist selected T cell populations (CD8αα(I), CD8αα(II), T(agonist), Treg(diff), and Treg) exhibited variable timing of induction. Expression profiles of chemokines and co-stimulatory molecules, together with spatial localisation, supported that dendritic cells, B cells, and stromal cells contribute to agonist selection, with different subsets influencing thymocytes at specific developmental stages within distinct spatial niches. Understanding factors influencing agonist T cells is needed to benefit from their immunoregulatory effects in clinical use.

Employment Growth and Industrial Policy: The Challenge for Indian States.

The nature of and the future potential for economic growth will vary across Indian States because of their differences in the rates of demographic transitions. The growing population of the young in some of the States in the east and north of the country, notably Bihar and Uttar Pradesh, opens up a huge economic opportunity. It is also a serious policy challenge-to create new opportunities that meet the rising expectations of the job aspirants. At the same time, for States such as Kerala and Tamil Nadu, which have an ageing population, there are limits to future growth based on labour-intensive sectors. Across Indian States, during the period between 2005 and 2018, labour absorption into industry, construction and services lagged way behind the increase in the potential supply of workers into these sectors. The mismatch between labour demand and potential labour supply widened after 2012, leading to an increase in unemployment rates and a large-scale withdrawal of women from the labour market. For reviving employment growth, India requires a mix of social, employment and industrial policies. The States should have greater financial and functional autonomy to implement these policies in a way that suit their specific stages of development.

Abstract A034: Orchestrated expression of the atypical Rho-GTPase, RHOV, in response to matrix detachment of ovarian cancer cells

Abstract Ovarian cancer (OVCA) metastasis occurs through the process of transcoelomic spread, where cells disseminate into the peritoneal fluid and form multi-cellular aggregates (MCA) that mediate Anchorage-Independent (A-I) survival and invasion of the peritoneal organs. Isolation of MCAs from patient ascites has been extensively reported in the literature and found to correlate with poor patient survival and resistance to therapy. However, the mechanisms promoting OVCA MCA formation and survival in A-I are not fully understood. We previously demonstrated that MCAs upregulate their mitochondrial antioxidant defenses to maintain optimal survival in A-I, and that this is an early event following cellular detachment. To identify additional key molecular players promoting MCA formation and fitness in A-I, we employed sequential RNA seq analysis to compare the transcriptome of cells in attached, early A-I, and late A-I conditions using the serous cell line OVCA433. Analysis of early transcriptomic changes observed within 2hrs of A-I revealed that RHOV, a recently identified atypical member of the Rho GTPase family, is the top gene significantly upregulated in early A-I. Moreover, we found that RHOV expression rapidly declines in later A-I timepoints indicative of a tight temporal regulation of RHOV expression following cellular detachment. Next, we compared the expression of RHOV in primary ovarian tumor cells and matching malignant ascites cells from the publicly available dataset (GEO: GSE85296) and found increased RHOV expression specifically in the metastatic ascites of ovarian cancer patients when compared to the attached tumor. Previously, RHOV expression was found to regulate intercellular adhesion dynamics during specific stages of neural crest development, a physiological process mimicking epithelial to mesenchymal transition (EMT). RHOV has also been reported to be overexpressed in lung cancer where it was found to correlate with poor patient outcome and resistance to therapy. However, the role of RHOV in ovarian cancer metastasis remains unexplored. To test the consequences of increased RHOV transcription in A-I: its role in migration, aggregate formation, and anoikis resistance is being tested following silencing of RHOV expression using both CRISPR and siRNA. Current work is exploring how RHOV is rapidly transcribed following matrix detachment and how increased expression alters cellular signaling events in early A-I stages to promote optimal MCA fitness. This work aims to contribute new knowledge on the novel oncogenic role of an understudied member of the Rho family of GTPases, RHOV. Moreover, given that increased MCA fitness has been correlated with increases metastatic potential and resistance to therapy, we provide a unique proof-of-concept study to target key components of early MCA adaptations as a novel therapeutic strategy for prolonging survival rates in OVCA patients diagnosed in metastatic stages. Citation Format: Amal T. Elhaw, Yeon Soo Kim, Zaineb Javed, Priscilla Tang, Weihua Pan, Nadine Hempel. Orchestrated expression of the atypical Rho-GTPase, RHOV, in response to matrix detachment of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A034.

How Memory Counts in Mathematical Development.

Memory has been well-established as a predictor of mathematics achievement in child development. Nevertheless, empirical evidence remains elusive on the unique role of the different forms of memory and their specific mechanisms as predictors of mathematics development. Therefore, in this study, the role of visuospatial short-term memory, visuospatial working memory, verbal short-term memory, and verbal long-term memory was investigated at three key stages of the development of mathematics (5-6 years, 6-7 years, 7-8 years), as well as their interactions across development. The relation between the different memory types and informal and formal mathematics was also studied. The findings of this study provide empirical support for a shift in the relation between different memory types and mathematics achievement over development with: 1) visuospatial short-term memory predicting informal mathematics achievement at the age of 5-6 years; 2) visuospatial working memory predicting informal and formal mathematics achievement at the age of 6-7 years; and 3) verbal short-term memory predicting formal mathematics achievement at the age of 7-8 years. These shifts clearly appear consistent with children's mathematics curriculum content over time and the requirements of mathematics acquisition at specific stages in development. With these findings, the unique role of various forms of memory in the development of mathematics and the timeframe in which they play a crucial part is highlighted, which should be taken into consideration for future research and possible intervention studies in children's mathematics achievement.

Unfolding a sequence of sensory influences and interactions in the development of functional brain laterality.

Evidence of sensory experience influencing the development of lateralized brain and behavior is reviewed. The epigenetic role of light exposure during two specific stages of embryonic development of precocial avian species is a particular focus of the research discussed. Two specific periods of light sensitivity (in early versus late incubation), each depending on different subcellular and cellular processes, affect lateralized behavior after hatching. Auditory and olfactory stimulation during embryonic development is also discussed with consideration of interactions with light-generated visual lateralization.

A toolkit for converting Gal4 into LexA and Flippase transgenes in Drosophila.

Drosophila has been a powerful model system for biological studies due to the wide range of genetic tools established for it. Among these tools, Gal4 is the most abundant, offering unparalleled tissue and developmental stage specificity for gene manipulation. In comparison, other genetic reagents are far fewer in choices. Here we present a genetic toolkit for converting Gal4 strains into LexA and Flippase transgenes through simple genetic crosses and fluorescence screening. We demonstrate the proof-of-principle by converting ten Gal4 lines that exhibit diverse tissue specificities and examined the activity patterns of the converted LexA and Flippase lines. Gal4-to-LexA and Flp conversion is fast and convenient and should greatly expand the choices of LexA and Flp for binary expression and FRT-based mosaic analysis, respectively, in Drosophila.

Floral Induction Systems for the Study of Arabidopsis Flower Development.

Assessing the molecular changes that occur over the course of flower development is hampered by difficulties in isolating sufficient amounts of floral tissue at specific developmental stages. This is especially problematic when investigating molecular events at early stages of Arabidopsis flower development, as floral buds are minute and are initiated sequentially so that a single flower on an inflorescence is at a given developmental stage. Moreover, young floral buds are hidden by older flowers, which presents an additional challenge for dissection. To circumvent these issues, floral induction systems that allow the simultaneous induction of a large number of flowers on the inflorescence of a single plant were developed. To allow the plant community to avail of the full benefits of these systems, we address some common problems that can be encountered when growing these plants and collecting floral buds for analysis.

An Insight into the Role of GLIS1 in Embryonic Development, iPSC Generation, and Cancer.

The curiosity to discover transcription factors to reprogram somatic cells to induced pluripotent stem cells (iPSCs) resulted in the identification of a reprogramming factor, Gli-similar transcription factor GLIS1. This proline-rich Kruppel-like zinc finger transcription factor has a role in embryonic development, iPSC generation, and cancer. The spatial and temporal expression of GLIS1 during embryonic development implicates that it can control gene expression at specific developmental stages. Moreover, GLIS1 in combination with OCT4, SOX2, and KLF4 reprogramming factors resulted in an increase in reprogramming efficiency, giving rise to primarily bona fide iPSCs. Mutations in the GLIS1 gene are associated with several types of tumors and cancers, and it shows a tissue-specific function where it acts either as an oncogene or as a tumor suppressor gene. This review gives a comprehensive overview of GLIS1 and its important role in embryonic development, cancer, and the generation of iPSCs.

Streamlined alpha‐amylase assays for wheat preharvest sprouting and late maturity alpha‐amylase detection

AbstractLate maturity alpha‐amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha‐amylase in wheat (Triticum aestivum L.) grain. Risk of poor end‐product quality due to elevated alpha‐amylase is detected in the wheat industry using the Hagberg–Perten falling number (FN) method. In breeding programs, selection for PHS and LMA tolerance requires higher throughput methods requiring a smaller sample size than the 7 g required for the FN method. Specifically, LMA can only be screened only using detection of alpha‐amylase activity or protein after cold treatment of individual wheat spikes at a specific stage of grain development resulting in very small samples (≤1 g). This study developed and evaluated a high throughput 96‐well method for the Phadebas alpha‐amylase enzyme assay for small wheat grain samples and compared this method to FN and the Megazyme Alpha‐Amylase SD (Sprout Damage) Assay Kit performed on the automated Awareness Technology ChemWell‐T Analyzer. In parallel, the efficacy of low‐cost small‐scale milling methods was evaluated relative to traditional larger scale mills. The Phadebas enzyme activity was highly reproducible and showed a strong correlation to the SD enzyme assay and FN method regardless of which mill was used to process the grain. The SD assay offers simpler standardization and calculation of enzyme activity, whereas the Phadebas assay offers higher sensitivity and lower expense. Both the 96‐well Phadebas and automated Megazyme SD assays are suitable for alpha‐amylase detection from small samples, and the use of low‐cost coffee grinders to process small samples did not significantly impact assay performance.